Dataset Information

Drosophila Sec16 mediates the biogenesis of tER sites upstream of Sar1 through an arginine-rich motif.

ABSTRACT: tER sites are specialized cup-shaped ER subdomains characterized by the focused budding of COPII vesicles. Sec16 has been proposed to be involved in the biogenesis of tER sites by binding to COPII coat components and clustering nascent-coated vesicles. Here, we show that Drosophila Sec16 (dSec16) acts instead as a tER scaffold upstream of the COPII machinery, including Sar1. We show that dSec16 is required for Sar1-GTP concentration to the tER sites where it recruits in turn the components of the COPII machinery to initiate coat assembly. Last, we show that the dSec16 domain required for its localization maps to an arginine-rich motif located in a nonconserved region. We propose a model in which dSec16 binds ER cups via its arginine-rich domain, interacts with Sar1-GTP that is generated on ER membrane by Sec12 and concentrates it in the ER cups where it initiates the formation of COPII vesicles, thus acting as a tER scaffold.

SUBMITTER: Ivan V

PROVIDER: S-EPMC2555954 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Drosophila Sec16 mediates the biogenesis of tER sites upstream of Sar1 through an arginine-rich motif.

Ivan Viorica V de Voer Gert G Xanthakis Despina D Spoorendonk Kirsten M KM Kondylis Vangelis V Rabouille Catherine C

Molecular biology of the cell 20080709 10

tER sites are specialized cup-shaped ER subdomains characterized by the focused budding of COPII vesicles. Sec16 has been proposed to be involved in the biogenesis of tER sites by binding to COPII coat components and clustering nascent-coated vesicles. Here, we show that Drosophila Sec16 (dSec16) acts instead as a tER scaffold upstream of the COPII machinery, including Sar1. We show that dSec16 is required for Sar1-GTP concentration to the tER sites where it recruits in turn the components of th ...[more]

PMID: 18614796

Similar Datasets

Project description:UnlabelledBacteriophage λ N protein binds boxB RNA hairpins in the nut (N utilization) sites of immediate early λ transcripts and interacts with host factors to suppress transcriptional termination at downstream terminators. In opposition to λ N, the Nun protein of HK022 binds the boxBs of coinfecting λ transcripts, interacts with a similar or identical set of host factors, and terminates transcription to suppress λ replication. Comparison of N-boxB and Nun-boxB nuclear magnetic resonance (NMR) structural models suggests similar interactions, though limited mutagenesis of Nun is available. Here, libraries of Nun's arginine-rich motif (ARM) were screened for the ability to exclude λ coinfection, and mutants were assayed for Nun termination with a boxB plasmid reporter system. Several Nun ARM residues appear to be immutable: Asp26, Arg28, Arg29, Arg32, Trp33, and Arg36. Asp26 and Trp33 appear to be unable to contact boxB and are not found at equivalent positions in λ N ARM. To understand if the requirement of Asp26, Trp33, and Arg36 indicated differences between HK022 Nun termination and λ N antitermination complexes, the same Nun libraries were fused to the activation domain of λ N and screened for clones able to complement N-deficient λ. Mutants were assayed for N antitermination. Surprisingly, Asp26 and Trp33 were still essential when Nun ARM was fused to N. Docking suggests that Nun ARM contacts a hydrophobic surface of the NusG carboxy-terminal domain containing residues necessary for Nun function. These findings indicate that Nun ARM relies on distinct contacts in its ternary complex and illustrate how protein-RNA recognition can evolve new regulatory functions.Importanceλ N protein interacts with host factors to allow λ nut-containing transcripts to elongate past termination signals. A competing bacteriophage, HK022, expresses Nun protein, which causes termination of λ nut transcripts. λ N and HK022 Nun use similar arginine-rich motifs (ARMs) to bind the same boxB RNAs in nut transcripts. Screening libraries of Nun ARM mutants, both in HK022 Nun and in a λ N fusion, revealed amino acids essential to Nun that could bind one or more host factors. Docking suggests that NusG, which is present in both Nun termination and N antitermination, is a plausible partner. These findings could help understand how transcription elongation is regulated and illustrate how subtle differences allow ARMs to evolve new regulatory functions.

Project description:BackgroundArginine Rich Motif (ARM) of HIV-1 Tat and Rev are extensively studied linear motifs (LMs). They are already established as an inefficient bipartite nuclear localisation signal (NLS). The unusual passive diffusion of HIV-1 NLS tagged reporter proteins across the nucleus is due to an unknown competing functionality of ARM. Recent findings about the role of retroviral proteins as a suppressor of RNA interference (RNAi) involving their basic residues hint an interesting answer to this alternate functionality. The present work explores the role of HIV-1 ARM as a uniquely evolved viral motif to combat Dicer dependent RNAi.ResultsWe show that RNA binding ARM of both HIV-1 Tat and Rev is a LM with a pattern RXXRRXRRR unique to viruses. Extending the in silico results to wet lab, we proved both HIV-1 Tat and Rev can suppress Dicer dependent RNA silencing process involving ARM. We show, HIV-1 Tat and Rev and their corresponding ARM can bind the RISC loading complex (RLC) components TRBP and PACT confirming ARM as an independent RNAi suppression motif. Enhancement of RNAi in infection scenario through enoxacin increases HIV-1 replication as indicated by p24 levels. Except Dicer, all other cytoplasmic RNAi components enhance HIV-1 replication, indicating crucial role of Dicer independent (Ago2 dependent) RNAi pathway in HIV-1 infection. Sequence and structural analysis of endo/exo-microRNA precursors known to be regulated in HIV-1 infection highlights differential features of microRNA biogenesis. One such set of miRNA is viral TAR encoded HIV-1-miR-TAR-5p (Tar1) and HIV-1-miR-TAR-3p (Tar2) that are known to be present throughout the HIV-1 life cycle. Our qPCR results showed that enoxacin increases Tar2 miRNA level which is interesting as Tar2 precursor shows Ago2 dependent processing features.ConclusionsWe establish HIV-1 ARM as a novel viral motif evolved to target the Dicer dependent RNAi pathway. The conservation of such motif in other viral proteins possibly explains the potent suppression of Dicer dependent RNAi. Our model argues that HIV-1 suppress the processing of siRNAs through inhibition of Dicer while at the same time manipulates the RNAi machinery to process miRNA involved in HIV-1 replication from Dicer independent pathways.

Dataset Information

Drosophila Sec16 mediates the biogenesis of tER sites upstream of Sar1 through an arginine-rich motif.

Publications

Drosophila Sec16 mediates the biogenesis of tER sites upstream of Sar1 through an arginine-rich motif.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets